Human kidney cancer cell surface antigen monoclonal antibody

[Detailed Description of the Invention]

The invention indicated especially is made with the research fault machine under the approval from the American government and a health education welfare prefecture, or a judgment. The present invention relates the generating Rice cake people renal cancer antigen of a monoclonal antibody to using the monoclonal antibody for that of identification and Characterize district. This is a diagnosis means effective in detection of kidney wAfIh, and research of the character of a renal cancer. Immunity fluorescence and 1 Oyo Enzyme Cinera agent are highly combinable with a unique antibody using an ordinary method which is made into the index method with 8 expenses. Familiarity which destroys alternatively the cell which combines a cytotoxic agent with a unique antibody highly, and the unique antibody combines "a bullet [ of magic ] (A gio bull-t) J type medical treatment agent is also generable. We are these days and mouse monoclonal antibodies (the analysis in early stages of [ about the cell surface antigen of people's malignant melanoma by which Beam bX Yo Si identification was carried out ] us was reported (Dippolcl et al.)). Proo, jail, Moe, Soi, upsilonS Beam, 7L6114-6118 (1111G), and the present invention relate to analysis of size Wisdom [8 which is equal to this. The monoclonal antibody of 1T guided from fusion into the Tile cell of the mouse by which immunity was carried out by the established cell line system of a renal cancer identified nine cell surface antigen systems. Six antigen systems (fP 1 a O, 15 ms, Can p12or, gps20nr.) gptts and Lt! It participates in other three antigen system t Yo HL muumuus showing the antigen which is not indicated until now, -B, -CH chain, Beam, and B blood group antigen. Thing Cao Cao gptso most limited among the newly indicated antigens, 81 arts and gp120r Some of these determinant sites are normal, and it is found out by only the cell of both malignant kidney origin, Besides the difference of zero molecular weight showing the judgment antigen of size Wisdom, they are these antigens, i.e., fpHio, So and gp120r O -- two -- To -- glycoprotein (glycoprotein) ticks which have *gp120r distinguishable based on the judgment expression on the panel of a cultivation renal cancer, a normal kidney epithelium, and an embryo kidney cell, Although kidney gP(by which it is shown by continuous sedimentation by monoclonal antibody which detects gplzor and [p120nr)120nr, and one determinant site are shared, gP120nr is found out by the cell kind of the wider range containing the cell line guided from a fibroblast, the ovary, a bladder, and colon cancer. Although the two remaining new antigen systems, i.e., gp 115, and vI have the feature of the judgment antigen which occurs in the wide range, they are distinguishable from gp120nr to Mutually again according to the difference of the judgment expression to the cell type of a molecular weight, heat stability (vI is a heat stability determinant site), and the another kind origin. It can inquire, while the character of a renal cancer is characterized using these systems. Therefore, different all and zetap1600 table j+ of cultivation of a renal cancer cell line and the normal kidney! If a model is compared, it will be distinguished from these two Ri with 4sigmamu. Laboratory of our Fool research of this stomach-cancer, and latest (Dippold and others, Proo, Natl, Beam oad, 801.upsilonS Beam, IT.) A series of mouse antigen Beam b which specify the system of 12 with a new person cell surface antigen from 11114-61Nl (198G) occurred. They are six pieces (identified as Yu glycoprotein (gP115+gP1sO, gPl& (1, fP120r.))) among these. the heat instability in which gP120nr and the gpttschi3 piece cannot carry out immunity sedimentation from a sign cell extract -- anti-1K (B proverb 1+Ml- and R-) -- Ah Si, The remaining three-piece Neck fever stable antigens and the thing which is probably glycolipids (glycolipid) (Os , Rta, and Vt) and for which it cultivates and uses the standard panel of a nine-person cell, Being able to measure easily the reactivity of these monoclonal antibodies in a direct serum test and absorption analysis, each antigen system presents the distribution pattern which was qualitative as for the antigen and clarified on the cell panel about both quantitive expression. It can classify into these antigen systems mu of 12, and also three groups based on the distribution on a different cellular type. (1) What has the feature of the limited judgment antigen (for example, gptso peculiar to Kidney society) A So and gP120r antigen and a melanoma, R14 antigen of a black blood cell, the judgment antigen (for example, gp952gp150.Mn 1gp120flr and V) in which (ll) was displayed widely suddenly, and the antigen expressed by the everybody thin fan type examined psi0 (for example, 0@ kind antigen). being guided from a stage Wll renal cancer (shut up by the kidney) -- nine cell lines -- gptso it is -- the cell line from a metastasis renal cancer -- gptso it is -- things were also confirmed. Impression that the cancer cell which this presents metastasis potentiality has lost gp160 expression. or suggesting that 1p160+ and a gptso-renal cancer are guided from another cell lineage -- Meeting -- laws -- Re -- 4 - there is nothing However, the information about the origin on the cell of a renal cancer should be acquired by Hitoshi who identifies Cytoplasm in the normal kidney which displays gp180 and other antigens which are found out by the renal cancer. These Serological Si by whom Yo Si offer is made at the present invention can identify an antigen peculiar to the kidney, and is [ research of a Kidney lies organization and 41I ability ] useful to 411. Moreover, some of antibodies which react to Mori extensively are useful although other serious injuries are studied, and Ah Si, for example, vI, distinguishes a stellate cell vagina from a melanoma. The average Tari biochemistry of an antigen and the importance of serological characterization which are identified by Beam b are shown by analysis of g120r and gP120nr in variant. If immunity sedimentation of the 120,000 Dalton ingredients is carried out from the extract with a sign of -RO-7 kidney tame A cow and reserve clearing of 9 and the extract is carried out to this 411JO5 o Beam bH113 by one of the Beam b of these (Mouret 8 No.), 120 The Beam bs1. identification ingredient of 000 Dalton was removed, and it suggested that these two OAb(s) had reacted to the same molecule. However, antigen Decided crystal detected by Beambs- and Beam ban is distinguishable by a M-MH whip strike and absorption analysis. An antigen peculiar to the kidney is detected, on the other hand, Beam b Ss+ Yo also reacts to the cell type of the wide range suddenly, and Beam bass is 9. These results "d'A [ The and ] by assuming 120 molecules of gp(s) of 24ji with which only one of the two supports the epitope identified by Beam l3□ although Engineering is Kawahata(ed) one time by Beam b B@ and Ruhetorp is supported. It agreed in this interpretation, and supernatant liquid after Furia knob(ing) by Beam bass still reacted to Beam bS6, although tJ: and the antigen carried out 14 did not remain by 5bSu. The epitope identified by Beam bfsll "it is found out by only the cell of - kidney origin and is called that limited distribution, therefore this epitope r-LIp120r . the epitope distributed more widely is displayed by rnr", in order to show the un-restrictive (non-re@ trio -- Corp.) character. Two separate genes or the product of those is a product of the single gene which denatures in a kidney cell, and gp12Lr and Kpl 20nr are Ah Si obtain. Also disagreement of cell distribution of the antigen identified by different monoclonal antibody which carries out immunity sedimentation of gpss or 150 molecules of the jp(s) although similarly it is remarkable and there is no r Yo than this, # ! -- lt iron is carried out -- truth -- it is explained based on an epitope (DIppOI(1 and others, Proo, Natl, Soi, US Beam, rr, 61j4-61111 (111110) ) e) Experiment aim cultivation. Renal cancer cell line (Ueda et al., field for J.) Mo2S, ISO, and 564-51111 (1979) De -- and the tumor cell system (Oarey* Proo, Natl, Moe, 8oi, IJ8 Beam, T3 *32111-32 and 2 (1117@)) u report of is done. Guru cumin of e 2am to which the short-term culturing method of the normal kidney epithelium has also been reported (Ueda (same as the above)), 1% of nonessential amino acid, Beni/Lynn of 100 units per 1 Scratchy, 9 that 1■L'' is supplemented with with streptomycin of Responsibility 4, and the new-born calf serum of 10 Chi (capacity/capacity), and maintains a cultivation stock at 9 eagle minimum essential media, A cultivation stock is regularly tested about a mycoplasma, a fungus, and bacteria (bacteria), a pollution cultivation stock is thrown away, and it is 9. Serological operation. Mouse mixture blood cell adsorption official approval (M-tH Beam) was corrected so that Yo Si and a mouse antibody might be detected to the method of hgr&5uJl and others (I■-1ogy, @, 1@1-175 (19@S)), and it was performed. The serological operation about a direct test and absorption analysis is indicated by Dippold et al. (same as the above), Ueda et al. (same as the above), C&xey and others (on iuJ). The immunity measures of immunization and (BBeamLB/c X C57BL/6) Ft female Max were taken by the stock-ized kidney Mh1111I A cow system (refer to the 1st table). Initial immunization "a hypodermic injection is carried out without a Horse mackerel- bunt of a Yu and I X 10' renal cancer cell, and it is 9. The intraperitoneal inoculation of the lXl0' kidney #S cancer cell performed subsequent immunity disposal by 4 for 3~4 weeks. The immunization mouse was sacrificed to after [ the last immunity disposal ] 3 Tachimori. Derivation of a mouse antibody (Beamb). About fusion in mouse myeloma MOPC[ of an immunity Tail lies cell ]-21NS/1 cell, they are Dippold et al. (same as the above) and Kohler & Milgtain. Mature (1, ormn) It carried out for having been indicated to 23G and 495-496 (1975), and passing. The fusion cell (5~8x10'') contained in selection j!! ground 1 Chill containing Hiboxanthi/, amino GUTILIN, and thymine /was added to Well of the Braided fiber cultivation plate (0ostsx no.). It is Yasu 4 well to 3524.1 plate #. By limiting the dilution on 1~3X10' one layer of feeders of the macrophage in ff A cup abdominal cavity, a A eve Lidoma (hybri, 6D Truth) cultivation stock is formed into a subclone 3 times or more, and he is a friend. Culture *m A cow which consists a cultivation supernatant fluid of two renal cancer cell lines (an immunization system is included), That is, they are -MXL-33, BK-MNL-31 melanoma, M Abuse-1110 cervix cancer, a vx-ssys child cell, a V Abuse RO adult, an embryo kidney epithelium, and embryo li to Beam JJIM Bladder and 8! It monitored about antibody activity on the panel of #l[l A cow. Antibody ?2 / 9JtKIg II chain unique trial @ (Bionsuos.) p determination of is done in a division and direct-neighborhood n, M, and D at double-among Martial arts diffusion, and it is 9. The culture of the Crowe y-ized hive Lidoma was m Carried out, and the mw Louthus background K hypodermic injection carried out, and serum was collected gradually [ 0 ] which was saved also in liquid nitrogen from the mouse in which the tumor grows, and it stored by -10 degreeC, and was serological, and used it for biochemical characterization. immunity sedimentation operation and a cell -- 1 -- Fenghua -- hit IS andQ [ C1 ] [1M] A -- 2 To i y (Nap XnglaaIINuolsar;30~11001AIIIDl) 101: Carry out sign attachment in a 48 hour generation-process by s1-degreeC by [8M] glucosamine in the perfect Eagle's medium containing x3.rXl 0"beoqu-rel, and it is 9. About a cell, they are Ogata et al., Proo, Natl, and Beam ov1.soi with mm. UfSA and 78.7TO-774(19111)le Loading 1t' -- it is carrying out -- as (a 3MKOL place at-saw outside carried out) -- O in taurijl buffer solution, and S % Mon11 i-tP-40(NP-40)-7' -- ?lIl took out. Some cell extracts (immunity sedimentation was performed by mixing with the mouse serum of 2 st in a 1x10'' Ofim t-Tris buffer solution, and the rabbit anti-1!81 A Ig serum (CtppelL& Seki 1 Love h and town Coo Fighting ule, P A) of 20microt.) a connoisseur given in DippoW and others (same as the above) -- Si, Using 5 Violent episode view rwg, it isolated and analyzed the immunity complex with the Na Dcxl 80 Mehopol acryl amide gel electrophoresis method. (81i5) immunity sedimentation of the Confidant attachment Was done sample was carried out depending on the opportunity or the method of Si by methionine. however, carrying out single [ of the complex ] -- S--side Gentleman It obtained and used point F (ab') and anti-mouse xC. In order to measure pI of an antigen, the immunity sedimentation thing was investigated by the two-dimensional electrophoresis by the 01 Ha n01 method (O''F "nl.). p, H, Blol, Chw, 250-4007-4021 (15175) The method of a statement is amended to Ogata and others (same as the above) like a statement. The antibody production clone of 1T was chosen as detailed analysis from five fusion which united Na-1 myeloma by three different renal cancer cell lines (the 1st table). The renal cancer of [13 which tested the serological singularity of these antibodies by the panel of an established cell line system of 41, the black haziness of 5, and neuroblastoma Gravity (gliaaas nauroblastcaw) , I The epithelial cancer of S, the Te cell line (MOL dish-4 and Ding-45) of BJiil fat systems 1562 (red leukemia erythroid l@ulcsta) and 2 of 5, and monkey kidney a A cow (VAbuseROl].) Zero person, the sheep, rat, and cow red corpuscles which tested these antibodies to short-term cultivation of a normal kidney epithelium, Embroidered leather gun, and an embryo organization (a brain, a fibroblast, and kidney -) were also investigated. In Okay., serological analysis was conducted by both the personal account test and the absorption test. Nine clear antigen systems were defined by these serological researches and immunization study analysis. Three systems (JinTh calling (D120r) 11>1@O, d it being normal and limited to a malignant Kidney training cell three systems (f120nr.)) gpHs and vI were widely distributed with Mori, and three systems were identified as HL muumuu, -B, -OH chain, Beam, and a B blood group antigen. Five Beam(b) of a ptgo antigen system and this series (5) at 160.000 which showed advanced singularity to * 8o e 5 proverb a k Yokohama MId person ffdja Border Dortono glycoprotein is identified. gp160 is a comparatively basic ingredient of pI7.s. By a M-Mii whip strike, gpi6Q can be proved about T of two and established cell line systems of 13 of a renal cancer of all the cultivation objects of a normal kidney epithelium, and the three cultivation objects of embryo kidney -, and he is a tiger. the -- 2# Ri and these results were checked by the absorption test. including the cell line guided from VKRO, i.e., the monkey kidney, -- other cellular type V Engineering -- neither [ all ] normal nor malignant were able to find out displaying gp180 antigen. 8 A and an antigen system. The antigen detected by Beam ban was also limited to the person cell of the kidney origin (Compilation 2 table). Although 811 determinant sites are heat instability and it has suggested that it consists on protein or mm confession, Beam bass did not settle an ingredient detectable from 8th division-Re-7 with [I"S] methionine Weaving or [8H] glucosamine Margin cell. 0 which will stretch in these two systems, will come and will be distinguished from Si if different culture CB mg of a renal cancer system and the normal kidney and gplso table 3j1wi are compared -- for example, -- calling 5 x-no-asp -- Moe 41aagptso / 811 Ah %ax-nc-6 -- gP1@0 It is /ams. nine cultures of a moreover and normal kidney epithelium -- + -- all -- fP1@0 Five of Ah Si and these cultures lacked '1-m one expression. gP120r and a gp120nr antigen system. Five Beam b (811-8 snow, -an-86, and 81) (am B) It [aH] Gurko [ methionine or ] t Came, immunity sedimentation of the 120.000-dollar Glycoprotein was carried out from alc-RO-7 with Reference thin 11i11 Dissolution, and the result same as the analysis under a circle, reduction nature, and nonreducible conditions was obtained. prototype Beam bas &! To Ab Bms Te identification Sale To gp1200pIu same Si c 4.@-! th2#O Beam b be [ nine 1.2 terrorism ] alike -- the result which Si identification is carried out and shows correlation of 120 ingredients 9 gp was obtained also from the continuous immunity sedimentation test. (" -- [) removing all the antigens which carry out preliminary treatment of the aK-RO-1 dissolution thing with a glucosamine sign by Beam ha- and which react to Yo 9 Beam b 8 ms especially -- a circle, Two things from which these gptzo antibodies distinguish 120 molecules of jp(s) of two groups, i.e., g120r, (limitation), and g120nr (un-limiting) and for which separate gP120 Engineering g Taupe was identified serologically were symmetrically shown by a M-MH whip strike and absorption analysis (the 2nd table). gp1'iox identified by Beam bSI has the distribution limited very much, and expressions are @ Had been to a normal kidney epithelium and a renal cancer of a certain kind. (gp120 epitope, i.e., gP120nr, of another side) It is carried out M constant by Beam b8@, and the e gP12or and gP120nr determinant site found out by the cultured cell of the wide range containing the cell line guided from an embryo and an adult Fiber blast cell, the ovary, a bladder, and colon cancer differs in expression by the renal cancer thin j1 system. 1i 5 K-RO-2 which all the cell lines do gp120nr epitope & possession of, -21, and -28 Si A week 0alC1-1 lack the II>12Or determinant site. The singularity of Beam bSn to the cell of the kidney origin is especially similar to the reactivity of Beam ban, and is gpi@. It is similar to the antibody which identifies A. However, gpts. and (in addition, 0 as which the antigen system peculiar to these three kidneys was chosen and which it can be normal or can be distinguished based on absorption analysis in a malignant kidney cell, for example, 81 m-RO-6, and 10 - p -- 120 -- an antigen -- a molecular weight -- a difference -- +) Moe 490 -- II>1@0 / Bus /gi12Or To!E. + The embryo kidney is ipl@Ot It was. It is A 8 proverb s"/gP120r-. gplIS antigen system. Beam bSn is under reduction or nonreducible conditions (immunity sedimentation of the 115.000 Dalton glycoprotein was carried out from -RG-7 cell lysate eight with "Ri glucosamine and ti ("8) methionine sign (Drawing 1)). In the direct M-MH whip strike, it turned out that high reactivity and (antibody titer 1~10.000X10-") are carried out in Si limited to the 15 mm of kidney 5rx Facial expression normal kidney epithelium ([2 table), and a galls antigen is expressed by various cellular types in absorption analysis. 9 did not carry out immunity sedimentation of the ingredient with a sign for v1 antigen system, Beam b v, Is, and ("II) glucosamine from 81 C-Re[ with [1@ Boss methionine Bamboo fiber ]-1 cell lysate. What an absorption test company antigen is heat stability (it is 5 minutes at 100 degreeC) is shown, and the product made from water of this antigen being a glycolipid (glycolipid) is carried out. * in which two features of v1 system (J112 table) are especially interesting (&) Although a V s system identifies the subset of a bladder, and a breast cancer is not vIf(ed) : expressed and -vI is not found out by Astrocytoma, melanomas are powerful V and @ expression object. that embryo guidance thing is related to Mutually at vI Contact -- 1 thin Breast tumor and a melanoma -- this -- stretching -- coming -- Seen from company does not have distinction carried out at other Beam b. HL Beam H strand. Beam b 8., Is (ssB) It is 45.Goo from a -RG-7 dissolution thing, and 12,000 to eight with a methionine sign. Immunity sedimentation of the Dalton ingredient was carried out. a direct test and an absorption test -- Beam b8 .. be alike -- the determinant site by which Si detection was carried out existed on the person cellular type of all Burned except for person red corpuscles (the 2nd table). the cell line which examines and does not react to Beam b8a with a direct MH whip strike in all nine-person cultured cells -- Ml-180 and 8 -MIL-110 -- By force -- four-person It was. -MIL-1@ evil spirit cell lines are HL muumuu from former research, and -B to 13. -Almost or completely not expressing O antigens is known. Beam bs, the ingredient O molecular weight come out and settled, and person! although Beam b8 and 1 detect HL Beam from the result of serological A matter of lll A cow -- H mirror top and beta1 Peng It turned out that the determinant site of a price increase is not distinguished. Fact d that person betal!n which isolated does not control the reactivity of Beam b 8s+, HL Beam The singularity about an H strand is suggested. In two of three renal cancer cell lines used for a Mus A week B blood group antigen and immunity (the 1st table), A fist ball+8 IC-ItO-7 which expresses Beam or B blood group antigen on the cell surface is B. 6J and 8 IC-RC-28+ are Beams. It is. BK-RO-fl is Casting(ed) from the individual of blood ff1O, and is positive at Beam and 3 reactivity. It is Beam in order to detect Beam b reacted to the antigen made from blood. The screen of the hive Lidoma supernatant fluid was carried out about the Blood clot S antibody using B, BeamB, or OWi red corpuscles. B condensation activity (Beam does not condense) is found out by four of the supernatant fluids of 4@2 from anti-5 K-RO-T (fusion) -- Beam condensation activity (B does not condense) -- anti- -- it was found out by 8 three of the supernatant fluids of 225 from -RO-2tl (fusion). Condensation of OWi red corpuscles is control test adult glycoprotein (5ullivan) of the 8th anti-division 3rd table AbM and, and Ab8.:blood type glycoprotein ' music. 1.3 120.0 person B glycoprotein (Beach) 150.0 1.2 person 0 (H) glycoprotein (Tighe) 246.0 123.0 Person Le" Glycoprotein (N-1) 475.0 237.0 Pig A10H Arthritis Matter 3.0 550.0 pig A gastric mucin (67) 4.6 437.0 pig H gastric mucin (66) -437, 0, 449, and 0* blood lid glycoprotein are provided by good will from E, A, and Kabat, and are indicated to (9). + 1 of condensation official approval by AII red corpuscles, and M, : 10,000 diluted-solution use. + 1 of condensation official approval by BIl red corpuscles, and 8. : 10,000 diluted-solution use. -R (found out by neither of the supernatant fluids, 3-7, -28, nor -6 (fusion).) Two single clone antibodies which have blood condensation ability from these fusion were guided. Beam and Beam b M to Beam B type red corpuscles. the blood condensation antibody titer of (nu/nu serum) -- 1.o-14 -- Off and B lid red corpuscles -- Beam b M -- it did not come out and condense. Beam b S to B and Beam B type red corpuscles (nu/nu serum) Although blood 'NIL condensation antibody titer was 4X10', Beam type red corpuscles were not condensed by Beam b 8-. To the 3rd table, they are Beam, B, H, and L ewis. The control test of Beam bS performed using the glycoprotein (Glycopectin) which has blood type reactivity, and a dead tree (Muhsin) extract, and Beam b M, is shown collectively. From this result to Beam b M The singularity and the B singularity of Beam b l are checked, and it is a circle. Applicant for a patent Sloanke and 1 TASTE GAINE STEEP r'r>-p7 Orr Cazooser Li +r-An agent Taki Hideko Depression A page [ 1st ] continuation Zero shot Ming person Bar Bert effort Gen America United States Connecticut new Canaan Opaltz The drive 48 Zero shot Ming '1 person Rye let F Witmore United States of America New York New York" and yeast 68 street 345 Zero shot Ming person jar shih tsu Kudraresh Poland 51-609 Urawla sergeous manusions 30 zero shot a Ming person Connie El fin -- a stud -- American New York New York yeast 81 Street * Nido 504 Zero shot - Tsubasa which a Ming person gives American New York New York yeast 81 street 345 Zero shot Ming person Ogata [ Toshikazu ] part Zero shot Ming person Ryuzo Ueda 1 * Kagokaya 1-12, Shimpo-cho, Chikusa-ku *'"2"0 -" j